A Clock-Driven Neural Network Critical for Arousal

Abstract

Main The function of WAKE is conserved in mammalsWe previously identified the clock-output molecule WIDE AWAKE (WAKE) from a forward genetic screen in Drosophila 4 . WAKE modulates the activity of arousalpromoting clock neurons at night, in order to promote sleep onset and quality 4,5 . The mammalian proteome contains a single ortholog, mWAKE (also named ANKFN1/Nmf9), with 56% sequence similarity and which is enriched in the core region of the master circadian pacemaker suprachiasmatic nucleus (SCN) 4,6 ( Fig. 1a, Extended Data Fig. 1a). To investigate whether the function of WAKE is conserved in mice, we generated a putative null allele of mWAKE (mWAKE (-) ) by CRISPR/Cas9 insertion of 8 base pairs (containing a stop codon and generating a downstream frameshift) in exon 4, which is predicted to be in all splice isoforms of mWAKE ( Fig. 1b). As expected, mWAKE expression, as assessed by quantitative PCR and in situ hybridization (ISH), was markedly reduced in mWAKE (-/-) mice, likely due to nonsense-mediated decay (Fig. 1c, 1d). Given mWAKE expression in the SCN, we first examined locomotor circadian rhythms and found that mWAKE (-/-) mice exhibit a mild but non-significant decrease in circadian period length (Extended Data Fig. 1b, 1c). These results are similar to findings from fly wake mutants and mice bearing the Nmf9 mutation (a previously identified ENU-generated allele of mWAKE) 4,6 .Because we previously demonstrated that WAKE mediates circadian regulation of sleep timing and quality in fruit flies 4,5 , we next assessed sleep in mWAKE (-/-) mice via electroencephalography (EEG). Under light:dark (L:D) conditions, there was no difference in the amount of wakefulness, non-rapid eye movement (NREM), or REM sleep between mWAKE (-/-) mutants and wild-type (WT) littermate controls (Extended Data Fig. 1d). In constant darkness (D:D), there is a modest main effect of genotype on wakefulness (P<0.05) and NREM sleep (P<0.05), and a mild but significant decrease in REM sleep in mWAKE (-/-) mutants (Fig. 1e). Although the amount of wakefulness did not appreciably differ in mWAKE (-/-) mutants compared to controls, there was a change in the distribution of wakefulness at night; mutants spent more daily time in prolonged wake bouts, and some mutants exhibited dramatically long bouts of wakefulness (Extended Data Fig. 1e, 1f).

Characterization of mWake expression in the murine brain

“…Wild-type (WT) mice in all experiments were C57BL/6J. WAKE Cre mice carry an insertion of a tdTomato-p2A-Cre cassette in exon 5 and was previously described (Bell et al, 2020).…”

“…A scRNA-Seq dataset was previously generated from flow-sorted tdTomato + cells of 7 week old male mWake (Cre/+) hypothalami (Bell et al, 2020) and used for analysis in this study (GSE146166). Briefly, an Act-Seq based protocol was used (Wu, Pan, Zuo, Li, & Hong, 2017) to dissociate the hypothalamus between the medial/lateral preoptic area and the beginning of the supramammillary nucleus.…”

“…Cells were flow-sorted based on tdTomato + fluorescence, and scRNA-Seq was then performed using the ×10 Genomics Chromium Single Cell system (v3.0 chemistry), following manufacturer's guidelines. Data were processed as described previously (Bell et al, 2020) using Seurat V3 (Stuart et al, 2019) to perform downstream analyses.…”

“…Our ongoing work on mWAKE suggests that the function of WAKE in regulating arousal by rhythmically modulating neural activity is conserved in mice. However, because mice are nocturnal animals, the increased arousal at night observed in mWake mutants manifests as hyperarousal, rather than changes in sleep (Bell et al, 2020).…”

Gene regulatory networks controlling differentiation, survival, and diversification of hypothalamic Lhx6-expressing GABAergic neurons

“…Seurat V3 (Stuart et al, 2019) was used to perform downstream analysis following the standard pipeline described previously (Bell et al, 2020), analyzing cells that express high Lhx6 transcript. Louvain algorithm was used to generate different clusters, and spatial information from individual clusters at E12.5 and E15.5 was identified by referring to our previous hypothalamus scRNA-Seq database HyDD (D. W. , as well as previous analysis of anatomical locations of transcription factors (Shimogori et al, 2010).…”